Project Summary Glucocorticoids (prednisone, methlyprednisolone, dexamethasone) have been employed for decades for control of hearing loss. However, little is known of the cellular and molecular processes of the ear that are under their control. Knowledge of these steroid-responsive mechanisms is critical for our understanding of normal cochlear function, as well as the design of appropriate clinical therapies. Therefore, the long term goal of this research is to fully characterize the steroid-driven cellular and molecular mechanisms of the ear. Progress on this study has shown that hearing loss in the MRL/MpJ-Faslpr autoimmune mouse responds to both the glucocorticoid prednisolone and the mineralocorticoid aldosterone. Furthermore, studies have shown glucocorticoids bind with equal affinity to both the glucocorticoid receptor and the mineralocorticoid receptor, thus regulating cochlear gene expression via immune suppression and ion homeostasis, respectively. Therefore, our working hypothesis is that two steroid-responsive mechanisms exist in the ear: a direct sodium and potassium transport (ion homeostatic) gene expression mediated by the mineralocorticoid receptor, and an indirect inflammatory gene suppression mechanism mediated by the glucocorticoid receptor. The planned studies will characterize these steroid driven cellular and molecular processes in the ear with steroid treatments that will functionally isolate the receptors and measuretheionhomeostaticand inflammatory gene expression they control. The specific aims to investigate these steroid driven mechanisms of the ear are: Aim 1: Determine the dose-dependent control of inner ear ion homeostatic and inflammatory gene expression by the mineralocorticoid aldosterone and the glucocorticoid prednisolone. Aim 2: Determine the most effective control of both inner ear ion homeostatic and inflammatory gene expression processes by combination doses of the two steroids. Aim 3: Determine which cochlear cellular and molecular functions are mediated by each steroid receptor. Aim 4: Determine if effective inner ear homeostatic and anti-inflammatory gene expression can be induced by middle ear steroid delivery. In all studies, 1) inner ear function will be assessed by auditory brainstem response audiometry and endocochlear potential; 2) inner ear morphology will be assessed by light and electron microscopy; 3) systemic autoimmune disease will be assessed by serum immune complexes, hematocrits, and antinuclear antibodies; 4) cochlearspecificautoantibodies willbeassessedwithELISA,and5)steroid-mediatedcochleargeneproducts will be assessed with ELISA, Western blot, cytokine RNA expression, and quantitative RT-PCR. The results from these studies will provide new findings regarding the cellular and molecular mechanisms of the ear that are under the control of steroids. This also will lay important groundwork for the potential development of steroid therapies more effective than those currently employed. Project Narrative A study is proposed to evaluate the mechanisms by which steroids control the maintenance of inner ear homeostasis. Studies are designed to better understand the role of glucocorticoids and mineralocorticoids in normal inner ear gene expression, and how steroid treatments may be operating to reverse hearing loss. Mouse models (MRL/lpr) with autoimmune inner ear disease, a steroid responsive disorder, will be employed to differentiate the ion homeostasis and immune suppressive functions of steroids and the efficacy of the two steroid classes combined. Finally, the functional impact of middle ear injections versus systemic delivery will be compared to determine the ability of each method to control glucocorticoid and mineralocorticoid receptor-mediated gene expression in the cochlea. Of particular interest are immune suppression of inflammatory cytokines and related transcription factors compared to the regulation of ion transport pathways that control fluid homeostasis.